PROJECT SUMMARY DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. Because ICLs are so toxic, many ICL-inducing agents are used as anticancer therapeutics. The use of such chemotherapeutic agents can also result in the formation of DNA-protein crosslinks (DPCs). While proteins from several repair pathways are thought to be involved in processing ICLs and DPCs, the mechanisms of ICL and DPC repair in mammalian cells are not clearly defined. Thus, the overall goal of the proposed studies is to fill this gap in knowledge by elucidating the mechanisms of crosslink repair in mammalian cells. We and others have demonstrated that proteins from several repair pathways are involved in ICL repair, including nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), Fanconi anemia (FA), and high mobility group (HMGB) proteins. However, their functions and interactions in ICL repair in mammalian cells are not well understood. The novel hypotheses to be tested are that: ERCC1-XPF is involved in the initial unhooking of ICLs in an SLX4- or UHRF1-dependent fashion; that HMGB1, but not HMGB3, is involved in the recruitment of ERCC1-XPF to ICLs to facilitate unhooking in an SLX4-independent fashion; and that ICL-induced DSBs are processed by mutagenic and error-free HR pathways, specifically regulated by HMGB proteins in mammalian cells. The long-term objectives are to elucidate molecular mechanisms involved in the removal of crosslinks from the mammalian genome. Specifically we propose to: (a) determine in vivo the mechanisms of incision and mutagenesis during crosslink repair; (b) elucidate the mechanisms involved in error-free and mutagenic crosslink repair associated with ICL- induced DSB formation; and (c) determine the mutagenic and recombinogenic impact of DPCs in human cells. The proposed work is innovative because it will test novel hypotheses; moreover, a unique feature of the approach is to induce site-specific ICLs in mammalian genomes to study their repair. In addition, genetically engineered mammalian cells lines constructed for this project will allow for definitive assessment of the roles of repair proteins in ICL processing in vivo, by separating the initial unhooking steps from the subsequent DSB- induced HR processing. These experiments will define the function of novel components (e.g. UHRF1 and HMGB proteins) involved in ICL repair, and have the potential to identify as yet undetermined proteins/pathways involved in the repair of crosslinks. The expected contribution is that the mechanisms of ICL and DPC repair in mammalian cells will be elucidated, which is significant because the new information obtained will aid in the development of novel targeted strategies to control human cancers using crosslinking agents. The studies will also contribute significant fundamental new knowledge to the fields of genetic instability and DNA damage and repair.